Enzymatic transesterification for biodiesel production

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A major economic disadvantage in chemical ... well, protein engineering has been used44. .... 8 Crabbe E, Nolasco-Hipolito C, Kobayashi G, Sonomoto K &.
Indian Journal of Biochemistry & Biophysics Vol. 40, December 2003, pp. 392-399

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Enzymatic transesterification for biodiesel production Shweta Shah, Shweta Sharma and M N Gupta* Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India Received 9 June 2003; revised 1 September 2003 Biodiesel consists monoalkyl esters of long chain fatty acids. It is produced from vegetable oils or fats either by chemical transesterification or by lipase-catalyzed transesterification with methanol or ethanol. Biodiesel is a green fuel and can be used as a blend with diesel or alone. Either way, it does not require any modification in engine design or storage facilities. The enzymatic process offers several advantages over the chemical routes. The handicap of increase in process cost because of the cost of the enzyme can be overcome by using efficient production process for enzyme and using reusable derivatives of enzymes, such as immobilized enzyme. Numerous strategies available in the area of non-aqueous enzymology can be exploited during the enzymatic alcoholysis for biodiesel production. Some of the technical challenges and their possible solutions are also discussed. Keywords: Biodiesel, transesterification reactions, lipases, oil-extraction, enzymes in organic solvents

Introduction The need for clean energy source is necessary because of the carcinogenic particulate emissions from diesel engines, which cause pollution and ‘global warming’. The depleting reserves of petroleum-based products have also made scientists look for renewable sources of energy. The concept of biodiesel addresses these twin issues. Biodiesel is an efficient, clean, nontoxic and biodegradable energy alternative to petroleum fuels1. Even though “diesel” is part of its name, there are no petroleum or other fossil fuels in biodiesel. “Biodiesel” means a monoalkyl ester that2: • is derived from domestically produced vegetable oils, renewable lipids, rendered animal fats or any combination of those ingredients; and • meets the requirements of ASTM PS121, the provisional specification for biodiesel. The ASTM standard3, Indian standard4 and standard D1NV516065 have been specified in the literature. In fact, the use of vegetable oils (e.g. from palm, soybean, sunflower, peanut, olive etc.) as such as alternative fuels for diesel engine dates back to almost a century6. The problem with direct use of such oils arise because of their higher viscosity and lower —————— *Author for correspondence Tel.: 91-11-26591503; 91-11-26596568 Fax: 91-11-26581073 Email: [email protected]

ignition quality as compared to diesel. The problems are more severe in the case of direct-injection engines than in the less efficient engines having precombustion engines. In the case of direct engines, very dilute blends of oils in diesel can be used6. Early approach to improve the usefulness of the vegetable oils consisted of pyrolysis of the oils. The pyrolysates had lower viscosities and higher cetane numbers than the parent vegetable oils7. Later work, of course, showed that the production of fatty acid methyl esters from the vegetable oils is a far more satisfactory approach. This conversion of the oil into the esters is essentially a transesterification reaction (Scheme 1). It is these fatty acid methyl esters or ethyl esters, which are collectively called biodiesel8. These transesterification reactions can be catalysed by acid or alkali8. Lately, considerable work has been carried out on production of biodiesel by using lipases as catalysts. The present review looks at these efforts after discussing the advantages associated with replacing chemical catalysts with enzymes in the context of biodiesel production. Sourcing starting material One of the two reactants in the transesterification is methanol or ethanol. As ethanol can be obtained from renewable sources, it is the preferred starting material. The degradation of starch by chemical or enzymatic method produces glucose; its fermentation by whole cell catalysis produces ethyl alcohol. At both steps, free as well as immobilized biocatalysts have been

SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION

Scheme 1—Biodiesel production from vegetable oils/fats by transesterification

used. The fat or oil can be obtained normally from seed or kernel from variety of plant sources. Unfortunately, there is a gap between the production and demand in the case of edible oil. Thus, a reasonable approach has been to search for oils, which are not used for edible purpose. Jatropha, a tropical plant has been extensively studied for production of biodiesel9. Its oil contains some toxic substances, which makes it unfit for human consumption. It may also be added that some people feel that using Jatropha oil for biodiesel production diverts it from its existing uses for soap making, lighting and motive power10. The oils from tobacco11, mango kernel12, palm8,13, soybean7,14, sunflower15,16, safflower17, cottonseed18, rapeseed19,20, Jatropha21 and beef tallow22 have been used for biodiesel production. Its preparations from waste or spent palm oil23 and restaurant grease24 are also worth mentioning. Shimada et al.25 have mentioned, “In Japan, 400000 tons of waste edible oils are discharged yearly. In this regard, several local governments in Japan have started collecting used frying oils from households and have converted them to biodiesel fuel for public transportation”. It is an example, which is worth emulating.

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Extraction of oil/fat from plant sources Generally, three main approaches for extraction of oil/fat have been used: hydraulic pressing, expeller pressing and solvent extraction26. While pressing and expelling can be used for oilseeds containing more than 35% oil, solvent extraction with hexane can be employed irrespective of the oil content27. However, the main problem with solvent extraction has been the production of volatile organic compounds, which harm the ecosystem. During the last several years, environmental concerns have led scientists to focus on development of methods, which do not utilize organic solvents. Aqueous oil extraction (AOE) consists of breaking the oil bodies mechanically in the presence of water and collecting the floating oil from the surface27. The yields of oil, understandably, are generally quite poor, but not insignificant. This encouraged the development of aqueous enzymatic oil extraction (AEOE), wherein enzymes like cellulase, hemicellulase and proteases are used to free oil bodies enmeshed in complex chemical structures by hydrolyzing the latter28,29. In our lab, AEOE has given high yields of 86 and 78% in the case of peanut and rice bran, respectively28,29. Recently, we have also described a novel approach of oil extraction by three-phase partitioning30. It was found that a mixture of salt and organic solvent mixed with homogenous plant material led to the formation of three phases: lower aqueous/water phase, interfacial precipitate containing mostly protein and upper organic phase, which contained oil. The oil can be recovered from the organic solvent by evaporation or freezing the mixture31. The technique has given promising results with oil from soybean30. Benefits of biodiesel In the past decade, biodiesel has been gaining worldwide popularity as an alternative energy source because of its many benefits. 1. Environmental benefits Biodiesel is the only alternative fuel in the US to complete EPA (Energy Policy Act) tier I health effects testing under section 211(b) of the clean air act, which provides the most thorough inventory of environmental and health effects attributes that current technology will allow32. Unlike other “clean fuels”, such as compressed natural gas (CNG), biodiesel and other biofuels are produced from renewable agricultural crops that assimilate carbon dioxide from the atmosphere to become plants and vegetable oil. Bio-

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diesel reduces the emission of carbon monoxide, ozone-forming hydrocarbons, hazardous diesel particulate, acid rain-causing sulphur dioxide, smoke and soot. It also lowers impact on marine environment by reducing toxicity32. 2. Biodiesel is the only alternative fuel that runs in any conventional, unmodified diesel engine. Also, it does not require any changes in storage facilities, which exist for storing petroleum-based diesel32. 3. Biodiesel can be used alone or mixed in any ratio with petroleum diesel fuel33. The most common blend is a mix of 20% biodiesel with 80% petroleum diesel, or “B20”. Biodiesel has positive performance attributes such as increased cetane number, high fuel lubricity, and high oxygen content, which may makes it a preferred blending stock with future ultra-clean diesel34. 4. Biodiesel is safe to handle and transport because it is “as biodegradable as sugar, 10 times less toxic than table salt”, and has a high flash point of about 300 F, compared to petroleum diesel fuel, which has a flash point of 125 F34. In fact, biodiesel is a proven fuel with over 30 million successful US road miles, and over 20 years of use in Europe32. It is the only alternative fuel that can actually extend engine life because of its superior lubricating properties34. 5. Energy security benefits Many countries, which have to import oil and other petroleum products become dependent upon other nations. Their economy, thus, is extremely vulnerable to any adverse effect of changes in political climate. Biodiesel, produced from domestic sources, generates self-reliance in a crucial area. Biodiesel production Biodiesel is produced from any fat or oil through a refinery process called transesterification (Scheme 1). Presently, the industrial production of biodiesel fuel is performed by alcoholysis of waste oil using alkaline catalysts. A by-product, glycerol, thus contains the alkali, and hence has to be treated as a waste material. Acid-catalysed transesterfication is another route for biodiesel production, which is more suitable for glycerides that have relatively high free fatty acid content and more water. Aksoy et al.35 reported that it was necessary to perform transesterification under an acidic condition when the oil composition was a lowgrade material containing sulphur etc.

Problems associated with chemical transesterification of vegetable oil • High reaction temperature36. • Soap formation due to the presence of free fatty acids in oil in case of base-catalyzed transesterification reduces the ester yields37. • A major economic disadvantage in chemical alcoholysis is that the purification of glycerol (produced as a secondary product) is very difficult16. • Homogeneous catalyst removed with glycerol layer cannot be reused. • For diesel fuel, ethyl ester is preferred because ethanol can be produced from biomass and is less toxic, but conventional alcoholysis with ethanol gives low yields21. In view of the above disadvantages associated with the chemical transesterification for biodiesel production, the enzymatic alcoholysis of oil/fat is considered desirable. Table 1 compares chemical and enzymebased methods for biodiesel production36. Enzymatic alcoholysis To overcome problems associated with chemical catalysis for production of biodiesel, enzymatic processes using lipases have been developed. Alcoholysis by lipases is considered one of the most effective reactions for the production of biodiesel. Lipases (E.C. 3.1.1.3) hydrolyze triglycerides and can be isolated from microorganisms, plants, mammals etc. The lipase-catalyzed reactions can be classified as follows38: (i) Hydrolysis R1COOR2 + H2O → R1COOH + R2OH Table 1—Enzymatic and non-enzymatic methods for biodiesel fuel production36 Conditions

Alkali-catalyzed

Lipase-catalyzed

Reaction temperature

60-70°C

30-40°C

Free fatty acids in raw materials

Saponified products

Methyl esters

Yields of methyl esters

Normal

Higher

Recovery of glycerol

Difficult

Easy

Purification of methyl esters

Repeated washing

None

Production cost of catalyst

Cheap

Relatively expensive

SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION

(ii) Synthesis: reactions under this category can be further divided: — Esterification R1COOH + R2OH → R1COOR2 + H2O — Transesterification Alcoholysis R1COOR2 + R3OH → R1COOR3 + R2OH Acidolysis R1COOR2 + R3COOH → R3COOR2 + R1COOH The summary of biodiesel production from various sources is given in Table 2. Biodiesel production, thus involves using enzymes in non-aqueous media. Non-aqueous enzymology and transesterification of oils and fat It is now well-established that the enzymes can be used under nearly anhydrous conditions of neat organic solvents as reaction media39,40. The enzymes under these conditions do require a minimum amount

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of water, which is less than monolayer amount of water molecules around the enzyme molecular surface. Enzymes, in fact, become highly thermostable and can be used at high temperatures. Lipases, for example have been found to be biologically active at 100°C in nearly anhydrous organic solvent. The major application of non-aqueous enzymology is synthesis by hydrolases. Thus, in the absence of water, thermodynamic reversibility pushes the enzyme-catalyzed reaction in the reverse gear. A major limitation in the use of enzymes in these media is their low catalytic efficiency. This has been improved by immobilization41,42, chemical modification43 and protein engineering44. For improving the catalytic efficiency as well, protein engineering has been used44. Recently, some workers have described ‘salt activation’ for improving catalytic efficiency45. Ultrasonication46 and microwave-irradiation47 have also been used in a limited way. The cost of the enzymes is, in fact, always a major deterrent in adopting their use in any biotechnological process. The two major approaches, which are viewed as possible partial solutions to the dilemma are: (i) efficient bioseparation strategies; and (ii) enzyme immobilization.

Table 2—Summary of biodiesel production by transesterification of plant oils/fats Route

Oil source

Catalyst

Alcohol

Chemical

Sunflower7

KOH

Methanol

96

Jatropha curcas21

KOH

Methanol

92

Crude palm11

H2SO4

Methanol

78

Soybean22

Candida rugosa

Methanol

30

Ethanol

Enzymatic (Free Enzyme)

Palm kernel

10

88.4

Psuedomonas cepacia

Methanol

Sunflower7

Psuedomonas fluorescens

Methanol Ethanol

82

Rapeseed16

Candida rugosa

2-ethyl-1-hexanol

97

Mucor mehei

C4-C18:1

86.8-99.2

Psuedomonas cepacia/

Ethanol

85.4

Mowrah,mango Kernel, Sal

19

Recycled 20

Enzymatic (Immobilized)

Yield (%)

Restaurant grease

Candida antarctica

Soybean13

Candida antarctica

Safflower

14

Cottonseed15

Methanol

97

Methanol

87.4

Psuedomonas fluorescens Candida antarctica

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, DECEMBER 2003

Efficient bioseparation strategies The major component of the production cost of any enzyme is generally the expenditure on the downstream processing operations48,49. With this realization, many novel bioseparation techniques have emerged as a more efficient and economical alterations. In classical downstream processing, a nonselective precipitation with salt, organic solvent or a water-soluble polymer (like PEG) is generally used to concentrate the enzyme solution. This is generally followed by a chromatographic step. The introduction of affinity chromatography as a final and polishing step has become de rigour in the last 2-3 decades48,49. This kind of protocol is still widely used and serves the purpose, if the enzyme is required in milligram amounts for research needs. For any large-scale application, it is necessary to follow somewhat different sequence48,49. One important paradigm shift has been to use affinity interactions in free solution50-52. Such an approach has been tried for lipases as well53. Using alginate as a smart macroaffinity ligand, it has been possible to selectively precipitate the lipase from Chromobacterium and porcine pancreas. The simple, scalable and one-step purification gave 1.7 and 5.6fold purification with 87 and 75% of the total activity recovered53. The other important factors like short process time, low cost of the macroaffinity ligand (alginate is cheap and can be recovered for reuse) and scalability makes this a valuable approach for obtaining adequately purified lipases for production of biodiesel. Another possible approach, which may be worth trying is the use of expanded bed chromatography48,49,54-56. The use of stable fluidized beds in this technique obviates the need for any pre-clarification or concentration step. If the chromatographic media uses an affinity ligand, affinity capture of lipases would be expected to give adequately pure enzyme. The use of these robust, economical and efficient processes and some others of similar nature has been reviewed earlier54-56. The adoption of these techniques is considered to be useful in cutting down the cost of downstream processing (and ultimately production costs) of enzymes. Immobilized enzymes42,51,57 Linking enzymes to insoluble matrices by a variety of methods (such as adsorption, covalent coupling and entrapment) provides several advantages. In numerous cases, the enzyme becomes more stable. In the

context of transesterification, when the media is predominantly non-aqueous, stability is a major issue. In aqueous media, where enzymes are freely soluble, immobilization provides with a means to recover the biocatalyst by centrifugation or filtration. In anhydrous media, enzyme molecules, though mostly insoluble, tend to clump together. Thus, immobilization is a good way of increasing the surface area of the biocatalyst. Bosley and Peilow58 have provided a critical review and necessary protocol for immobilization of lipases. Their recommendation includes the use of celite or AccurelTM (macroporous polypropylene). A protocol for lipase immobilization by sol-gel technique is also described. It is also possible to extensively cross-link the enzyme to obtain insoluble chemical aggregates, which often show the advantage of enhanced stability and reusability without increasing the required reaction volume (as these do not have any volume occupying carrier). A recent innovation in this area is CLECTM (cross-linked enzyme crystal) developed by Altus biologicals (USA)59. CLECTM form of lipases from Pseudomonas cepacia (CLECPC) and Candida rugosa (CLEC-CR) are also available60,61. Effect of presence of solvent on biodiesel production Biodiesel production using enzymes from oil and alcohol has been tried in the presence and absence of solvent. Methanolysis of tallow oil, using Mucor mehei lipase in hexane has led to 77.8% ester yield19. Lately, solvent-free transesterification reaction is favoured by many workers, since it is more economical. Also, the toxicity and flammability of organic solvents, and product recovery without further organic solvent evaporation favours the use of solvent-free reaction62. Öznur et al.18 reported alcoholysis of cotton seed oil in a solvent-free medium, using immobilized Candida antarctica lipase with 92% of the total ester yield. The use of Rhizopus oryzae lipase (whole cell biocatalyst) for the synthesis of methyl esters (MEs) in a solvent-free system led to 71% MEs after 165 hr reaction at 37°C with stepwise addition of methanol63. Table 3 lists and compares the ester yields obtained by alcoholysis with various lipases in the presence and absence of solvent. For alkali-catalyzed transesterification, the glycerides and alcohol must be substantially anhydrous because water produces soap. The soap reduces the catalytic efficiency by consuming the catalyst and, increases viscosity, gelation,

SHAH et al.: ENZYMATIC TRANSESTERIFICATION FOR BIODIESEL PRODUCTION

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Table 3—Effect of presence and absence of solvent on enzyme-based biodiesel production Alcohol

Oil source

Solvent

Lipase

Yield

References

Methanol

Tallow

Hexane

Mucor miehei

94.8

16

Tallow

None

Mucor miehei

19.4

16

Cotton seed

None

C. antarctica

92

15

Restaurant grease

None

Thermomyces lanuginosa

4

20

Restaurant grease

None

C. antarctica (immobilized)

27

20

Tallow

Hexane

Mucor miehei

98

16

Tallow

None

Mucor miehei

65.5

16

Restaurant grease

None

Thermomyces lanuginosa

87

20

(immobilized)

Ethanol

(immobilized)

Propanol

Isopropanol

Restaurant grease

None

C. antarctica (immobilized)

76

20

Restaurant grease

None

Thermomyces lanuginosa (immobilized)

87

20

Restaurant grease

None

C. antarctica (immobilized)

79

20

Tallow

Hexane

C. antarctica

51.7

16

Tallow

None

Mucor miehei

90.3

16

Restaurant grease

None

Thermomyces lanuginosa

61

20

(immobilized)

Butanol

Restaurant grease

None

C. antarctica (immobilized)

59

20

Restaurant grease

None

Thermomyces lanuginosa

90

20

Restaurant grease

None

C. antarctica (immobilized)

56

20

Tallow

Hexane

Mucor miehei

83.8

16

Tallow

None

Mucor miehei

96.4

16

Restaurant grease

None

Thermomyces lanuginosa

97

20

89

20

(immobilized)

2-Butanol

(immobilized) Restaurant grease

None

C. antarctica (immobilized)

thereby causing difficulty in glycerol separation. Nelson et al.19 have reported the solvent-free lipasecatalyzed methanolysis of oils and fats to be sensitive to water. In contrast, Hsu et al.64 reported water activity (aw) to be an important parameter for methyl ester synthesis. Water activity